Calcium Homeostasis
Calcium homeostasis is the mechanism by which the body maintains adequate calcium levels. The process is highly regulated, and involves a complex interplay between calcium absorption, transport, storage in bones, deposition in other tissues, and excretion.
PTH is the most important regulator of serum calcium levels, and functions to increase the concentration of calcium in the blood by enhancing the release of calcium from bone through the process of bone resorption; increasing reabsorption of calcium from the renal tubules; and enhancing calcium absorption in the intestine by increasing the production of 1,25-(OH)2 vitamin D, the active form of vitamin D. PTH also stimulates phosphorus excretion from the kidney, and increases release from bone.
PTH secretion is regulated by the calcium sensing receptor (CaSR), a G-protein coupled receptor expressed on the cell surface of parathyroid cells, which detects small fluctuations in the concentration of extracellular calcium ion (Ca2+) and responds by altering the secretion of PTH. Activation of the CaSR by Ca2+ inhibits PTH secretion within seconds to minutes, and this process may be modulated by protein kinase C (PKC) phosphorylation of the receptor. The CaSR is also expressed on osteoblasts and in the kidney, where it regulates renal Ca2+ excretion.
In addition, PTH regulates phosphorus homeostasis. PTH stimulates the parathyroid hormone receptor 1 (PTHR1) on both apical (brush border membrane) and basolateral membranes. PTHR1 stimulation leads to an increase in urinary excretion of phosphate (Pi) as a consequence of reduction by internalization of the renal Na+/phosphate (NaPi-IIa) co-transporter on the brush border membrane. PKC activation would be expected to similarly reduce Pi excretion.
PTH is also involved in the regulation of osteoblasts and osteoclasts in bone. PTH increases serum Ca2+ by increasing bone resorption and renal absorption of calcium. PTH stimulates osteoblasts to produce RANK ligand (RANKL), which binds to the RANK receptor and activates the osteoclasts, leading to an increase in bone resorption and an increase in serum Ca2+. Osteoprotegerin (OPG) is a decoy receptor for RANKL which blocks bone resorption. Osteoporosis is caused by an imbalance between the processes of bone resorption by osteoclasts and bone formation by osteoblasts.
Hypercalcemia and Hyperparathyroidism
The human body contains approximately 1 kg of calcium, 99% of which resides in bone. Under normal conditions, circulating calcium ion (Ca2+) is tightly maintained at a level of about 8 to 10 mg/dL (i.e., 2.25-2.5 mmol/L; ˜600 mg). Approximately 1 g of elemental calcium (Ca2+) is ingested daily. Of this amount, approximately 200 mg/day is absorbed, and 800 mg/day is excreted. In addition, approximately 500 mg/day is released by bone resorption or is deposited into bone. About 10 g of Ca2+ is filtered through the kidney per day, with about 200 mg appearing in the urine, and the remainder being reabsorbed.
Hypercalcemia is an elevated calcium level in the blood. Acute hypercalcemia can result in gastrointestinal (anorexia, nausea, vomiting); renal (polyuria, polydipsia), neuro-muscular (depression, confusion, stupor, coma) and cardiac (bradycardia, first degree atrioventricular) symptoms. Chronic hypercalcemia is also associated with gastrointestinal (dyspepsia, constipation, pancreatitis); renal (nephrolithiasis, nephrocalcinosis), neuro-muscular (weakness) and cardiac (hypertension block, digitalis sensitivity) symptoms. Abnormal heart rhythms can result, and EKG findings of a short QT interval and a widened T wave suggest hypercalcemia. Hypercalcemia may be asymptomatic, with symptoms more commonly occurring at high calcium levels (12.0 mg/dL or 3 mmol/l). Severe hypercalcemia (above 15-16 mg/dL or 3.75-4 mmol/l) is considered a medical emergency: at these levels, coma and cardiac arrest can result.
Hypercalcemia is frequently caused by hyperparathyroidism, leading to excess bone resorption and elevated levels of serum calcium. In primary sporadic hyperparathyroidism, PTH is overproduced by a single parathyroid adenoma; less commonly, multiple adenomas or diffuse parathyroid gland hyperplasia may be causative. Increased PTH secretion leads to a net increase in bone resorption, with release of Ca2+ and phosphate (Pi). PTH also enhances renal reabsorption of Ca2+ and inhibits reabsorption of phosphate (Pi), resulting in a net increase in serum calcium and a decrease in phosphate.
Secondary hyperparathyroidism occurs when a decrease in the plasma Ca2+ level stimulates PTH secretion. The most important cause of secondary hyperparathyroidism is chronic renal insufficiency, such as that in renal polycystic disease or chronic pyelonephritis, or chronic renal failure, such as that in hemodialysis patients. Excess PTH may be produced in response to hypocalcemia resulting from low calcium intake, GI disorders, renal insufficiency, vitamin D deficiency, and renal hypercalciuria. Tertiary hyperparathyroidism may occur after a long period of secondary hyperparathyroidism and hypercalcemia.
Malignancy is the most common cause of non-PTH mediated hypercalcemia. Hypercalcemia of malignancy, is an uncommon but severe complication of cancer, affecting between 10% and 20% of cancer patients, and may occur with both solid tumors and leukemia. The condition has an abrupt onset and has a very poor prognosis, with a median survival of only six weeks. Growth factors (GF) regulate the production of parathyroid hormone-related protein (PTHrP) in tumor cells. Tumor cells may be stimulated by autocrine GF to increase production of PTHrP, leading to enhanced bone resorption. Tumor cells metastatic to bone may also secrete PTHrP, which can resorb bone and release additional GF which in turn act in a paracrine manner to further enhance PTHrP production.
Calcimimetic Agents
Calcimimetic agents are drugs that mimic the action of calcium on various tissues. Phenylalkylamine calcimimetic agents with activity on the parathyroid calcium sensing receptor (CaSR) have been described. See Nemeth et al., Proc. Natl. Acad. Sci., 95:4040-4045 (1998). One such agent, Cinacalcet, is marketed for the treatment of hyperparathyroidism. In addition, the CaSR can also sense and respond to other divalent and polyvalent cations, and to organic polycations, such as spermine, hexacyclin, polylysine, polyarginine, protamine, amyloid β-peptides, neomycin, and gentamycin, although these cations are reported to lack selectivity and to possess relatively low potency for the CaSR. See Nagano & Nemeth, J. Pharmacol. Sci., 97:355-360 (2005); see also Brown et al., J. Bone Miner. Res., 6:1217-1225 (1991).
Protein Kinase C
Protein kinase C (PKC) is a key enzyme in signal transduction involved in a variety of cellular functions, including cell growth, regulation of gene expression and ion channel activity. The PKC family of isozymes includes at least 11 different protein kinases which can be divided into at least three subfamilies based on their homology and sensitivity to activators. Members of the classical or cPKC subfamily, alpha, beta (βI, βII), and gamma isozymes, contain four homologous domains (C1, C2, C3 and C4) inter-spaced with isozyme-unique (variable or V) regions, and require calcium, phosphatidylserine (PS), and diacylglycerol (DG) or phorbol esters for activation. Members of the novel or nPKC subfamily, delta, epsilon, eta, and theta isozymes, do not require calcium for activation. Finally, members of the atypical or aPKC subfamily, zeta and lambda/iota isozymes, are insensitive to DG, phorbol esters and calcium.
Studies on the subcellular distribution of PKC isozymes demonstrate that activation of PKC results in its redistribution in the cells (also termed translocation), such that activated PKC isozymes associate with the plasma membrane, cytoskeletal elements, nuclei, and other subcellular compartments. It appears that the unique cellular functions of different PKC isozymes are determined by their subcellular location. For example, activated βI PKC is found inside the nucleus, whereas activated βII PKC is found at the perinucleus and cell periphery of cardiac myocytes. Further, in the same cells, epsilon PKC binds to cross-striated structures (possibly the contractile elements) and cell-cell contacts following activation or after addition of exogenous activated epsilon PKC to fixed cells. The localization of different PKC isozymes to different areas of the cell in turn appears due to binding of the activated isozymes to specific anchoring molecules termed Receptors for Activated C-Kinase (RACKs).
RACKs are thought to function by selectively anchoring activated PKC isozymes to their respective subcellular sites. RACKs bind only activated PKC and are not necessarily substrates of the enzyme. Nor is the binding to RACKs mediated via the catalytic domain of the kinase. Translocation of PKC reflects binding of the activated enzyme to RACKs anchored to the cell particulate fraction and the binding to RACKs is required for PKC to produce its cellular responses. Inhibition of PKC binding to RACKs in vivo inhibits PKC translocation and PKC-mediated function.
cDNA clones encoding RACK1 and RACK2 have been identified. Both are homologs of the beta subunit of G proteins, a receptor for another translocating protein kinase, the beta-adrenergic receptor kinase, beta-ARK. Similar to G-proteins, RACK1, and RACK2 have seven WD40 repeats. Recent data suggest that RACK1 is a selective anchoring protein for activated βII PKC. Studies have shown that RACK2 (also called β′-COP) is a selective binding protein for εPKC. Csukai et al. J. Biol. Chem. 1997; 272:29200-29206.
Translocation of PKC is required for proper function of PKC isozymes. Peptides that mimic either the PKC-binding site on RACKs or the RACK-binding site on PKC are isozyme-specific translocation inhibitors of PKC that selectively inhibit the function of the enzyme in vivo.